Palmitoylation of KSHV pORF55 is required for Golgi localization and efficient progeny virion production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus etiologically associated with multiple malignancies. Both latency and sporadic lytic reactivation contribute to KSHV-associated malignancies, however, the specific roles of many KSHV lytic gene products in KSHV replication remains elusive. In this study, we report that ablation of ORF55, a late gene encoding a tegument protein, does not impact KSHV lytic reactivation but significantly reduces the production of progeny virions. We found that cysteine 10 and 11 (C10 and C11) of pORF55 are palmitoylated, and the palmytoilation is essential for its Golgi localization and secondary envelope formation. Palmitoylation-defective pORF55 mutants are unstable and undergo proteasomal degradation. Notably, introduction of a putative Golgi localization sequence to these palmitoylation-defective pORF55 mutants restores Golgi localization and fully reinstates KSHV progeny virion production. Together, our study provides new insight into the critical role of pORF55 palmitoylation in KSHV progeny virion production and offers potential therapeutic targets for the treatment of related malignancies. Author summary: Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple human malignancies. Nonetheless, the roles of numerous viral proteins in the viral life cycle remain inadequately characterized. Employing CRISPR knockout screening, we identified the viral tegument protein pORF55 as pivotal in the production of infectious progeny virions. We found that pORF55 is palmitoylated at cysteine 10 and 11, which is required for its Golgi localization. Palmitoylation-deficient mutants of pORF55 are unstable and fail to support secondary envelopment formation, a critical step for viral assembly and egress. Interestingly, we found that forced restoration of the Golgi localization of the palmitoylation-deficient pORF55 mutants completely reinstates the infectious progeny virion production. Hence, our study underscores the central role of Golgi localization resulting from pORF55 palmitoylation. Our study not only elucidates the role of pORF55 in viral replication, but also suggests targeting its palmitoylation as a potential therapeutic strategy for curtailing viral replication and treating related pathogenesis. [ABSTRACT FROM AUTHOR]